Proteins used for the diagnosis of lyme borreliosis

ABSTRACT

Chimera proteins including: (i) at least one sequence of a VlsE protein of a  Borrelia  species selected from  B. garinii, B. burgdorferi sensu stricto  and  B. afzelii , and (ii) at least one sequence including sequences of the IR6 domain of  B. burgdorferi sensu stricto, B. afzelii  and  B. garinii . Also, a method and a kit for the in vitro diagnosis of Lyme borreliosis using said proteins.

Lyme borreliosis (LB) is a noncontagious infectious disease caused by aspirochete called Borrelia burgdorferi, which is transmitted to humansvia a bite by a tick of the genus Ixodes. Without treatment, LB leads tovarious pathological disorders (dermatological, arthritic, cardiac,neurological and sometimes ocular disorders). It is the most commonvector-borne disease in the USA and in certain temperate countries ofthe northern hemisphere.

Several borrelia species, currently denoted under the group termburgdorferi or Borrelia burgdorferi sensu lato (including Borreliaburgdorferi sensu stricto, B. garinii and B. afzelii), are involved inthis infection. These species are pathogenic to humans.

In the United States, the infectious species involved is Borreliaburgdorferi sensu stricto. In Europe, in addition to this species, B.garinii and B. afzelii are involved. In Asia, the species involved areB. garinii and B. afzelii.

In the United States, approximately 10 000 cases are reported. InEurope, the incidence rates vary from less than 5 per 100 000.

Lyme borreliosis progresses by passing through three distinct phases,from early infection to the late phase. The early stage (stage I) may beasymptomatic or reflected by flu-like symptoms. In 50-80% of cases, theappearance of an inflammatory skin rash with a very particularappearance, called erythema migrans (EM) is noted several days after thebite by the tick. In the absence of treatment, the dissemination of theBorrelia via the blood is reflected a few weeks later by the occurrenceof inflammatory arthritis, neurological (neuroborreliosis) and meningealinvolvement, and skin and cardiac manifestations (stage II). Afterseveral months or years, the disease progresses to a chronic atrophicansform, encephalopathy, encephalomyelitis and chronic arthritis (stageIII).

A particular organotropism exists for each of the species of Borreliaburgdorferi. While the first stage of erythema migrans is withoutdistinction linked to the three species, the progression to aneurological form is preferentially associated with the species B.garinii, arthritis is more associated with B. burgdorferi sensu stricto,and acrodermatitis chronica atrophicans is specific for B. afzelii.

The similarity of the clinical symptoms between Lyme borreliosis andother unrelated diseases, and also the variability in manifestations,makes clinical diagnosis difficult. The diagnosis of borreliosis can beparticularly difficult on the basis of clinical observations, if casehistory evidence is absent (tick bite or EM). The early stage of thedisease may be without visible symptoms up to the time it reaches veryadvanced clinical stages.

Consequently, the diagnosis of LB is based on clinical signs but also onthe detection of pathogenic Borrelia burgdorferi-specific antibodies inthe serum, most commonly by ELISA (Enzyme Linked ImmunoSorbent Assay) orelse EIA or IFA.

In Europe, the evaluation of the serological response is complicatedowing to the existence of three pathogenic species and to theinterspecies variability for the major immunodominant antigens. Theantigens currently routinely used for detecting LB IgGs and IgMs areultrasound-treated cell samples of Borrelia burgdorferi sensu lato. Theperformance levels of the serological assays with these antigens, interms of specificity and sensitivity, are highly variable. Thus, owingto insufficient specificity, involving cross reactivities withantibodies associated with pathogenic bacteria, in particular Treponemapallidum (etiological agent for syphilis), spirochetes, rickettsiae,ehrlichia, or Helicobacter pylori, the diagnosis of samples havingtested positive by ELISA must be confirmed by immunoblotting.Sensitivity is also a major factor. This is because Borrelia burgdorferisensu lato expresses various surface proteins via adaptation to variousmicroenvironments, such that the genetic diversity and the differentialexpression of the Borrelia burgdorferi genes in patients have importantimplications for the development of serological tests for LB.

It was therefore necessary to develop a kit which overcomes theabovementioned drawbacks and which more particularly meets the expectedspecificity and sensitivity criteria.

The VlsE protein (surface expressed lipoprotein with Extensive antigenicVariation) is mainly expressed, in vivo, transiently and rapidly afterinfection of the host. It is very immunogenic in the infected host,involving the production of IgGs and IgMs. The Vls locus is located on alinear plasmid of 28 kb (Ip28-1) present in the three Borreliagenospecies responsible for Lyme disease and composed of silentcassettes and an expression site (VlsE). In vivo, random recombinationsbetween expression cassettes and silent cassettes occur during infectionand are responsible for the antigenic variability of VlsE. The VlsEprotein is composed of six variable regions VR1-VR6, located at thesurface of the VlsE protein, spaced out by “invariable” regions IR1-IR6.

It is known that the VlsE proteins exhibit considerable interspecies andintraspecies heterogeneity. In 2004, Göttner et al. [1] described anidentity of approximately 47 to 58% at the protein level of VlsEoriginating from four strains.

In order to overcome the abovementioned sensitivity and specificityproblems, the inventors have produced a Borrelia chimeric proteincomprising at least one sequence of the extracellular domain of a VlsEprotein of a first Borrelia species corresponding to a predeterminedstrain and at least one sequence of an IR6 region of a VlsE protein of asecond Borrelia species or of the first Borrelia species butcorresponding to a strain different than that of the first species, saidchimeric protein comprising (or consisting essentially of or elseconsisting of):

-   -   the sequence of the extracellular domain of the VlsE protein of        the first Borrelia species which is composed of five variable        regions VR1, VR2, VR3, VR4 and VR5 and of six invariable regions        IR1, IR2, IR3, IR4, IR5 and IR6, said at least one sequence of        the extracellular domain being selected from the group        consisting of SEQ ID NOs: 1, 2, 3, 4 and 5 or a variant of one        of said sequences SEQ ID NOs 1, 2, 3, 4 and 5, said variant        exhibiting at least 50% identity (preferably at least 60% or at        least 70% identity and advantageously at least 80% or at least        85% identity) with SEQ ID NOs 1, 2, 3, 4 and 5, respectively, on        the condition that said variant is capable of forming an        immunological complex with antibodies produced following a        Borrelia infection, and    -   the at least one sequence of the IR6 region of the second        Borrelia species, or of the first Borrelia species but        corresponding to a strain different than that of the first        species, which is selected from the group consisting of SEQ ID        NOs: 6, 7 and 8 or a variant of one of said sequences SEQ ID NOs        6, 7 and 8, said variant exhibiting at least 80% identity        (preferably at least 85% and advantageously at least 90%        identity) with SEQ ID NOs 6, 7 and 8, respectively, on the        condition that the variant of said sequence is capable of        forming an immunological complex with the antibodies produced        following a Borrelia infection.

The chimeric protein identified above can in addition comprise avariable sequence VR6 of a Borrelia species, this sequence beingidentified in SEQ ID NO: 9 in the sequence listing.

A preferred chimera protein comprises (or consists essentially of orconsists of):

-   -   the sequence SEQ ID NO: 1 or a variant of said sequence SEQ ID        NO: 1, said variant exhibiting at least 50% identity (preferably        at least 60% or at least 70% identity and advantageously at        least 80% or at least 85% identity) with SEQ ID NO: 1,    -   the sequence SEQ ID NO: 6 or a variant of said sequence SEQ ID        NO: 6, said variant exhibiting at least 80% identity (preferably        at least 85% and advantageously at least 90% identity) with SEQ        ID NO: 6,    -   the sequence SEQ ID NO: 7 or a variant of said sequence SEQ ID        NO: 7, said variant exhibiting at least 80% identity (preferably        at least 85% and advantageously at least 90% identity) with SEQ        ID NO: 7, and    -   the sequence SEQ ID NO: 8 or a variant of said sequence SEQ ID        NO: 8, said variant exhibiting at least 80% identity (preferably        at least 85% and advantageously at least 90% identity) with SEQ        ID NO: 8,    -   and, optionally, the variable sequence VR6 identified in SEQ ID        NO: 9.

Thus, one of the chimeric proteins of the invention comprises (orconsists essentially of or consists of) the sequence SEQ ID NO: 1, thesequence SEQ ID NO: 6, the sequence SEQ ID NO: 7 and the sequence SEQ IDNO: 8, or even in addition the sequence SEQ ID NO: 9.

The preferred chimeric proteins of the invention are particularlyidentified as comprising (or consisting essentially of or consisting of)a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 23;the most preferred protein being that which comprises or which consistsof a sequence identified in SEQ ID NO: 20 in the sequence listing.

SEQ ID NO: 1 corresponds to the sequence of the VlsE extracellulardomain of B. garinii (strain pBi) deleted of its signal sequence (aa1-19) and of the C-terminal region of the mature protein located afterthe IR6 domain, i.e. this extracellular domain is composed of the IR1,VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6 regions of B.garinii (strain pBi).

SEQ ID NO: 2 corresponds to the sequence of the VlsE extracellulardomain of B. garinii (strain pBr) deleted of its signal sequence and ofthe C-terminal region of the mature protein located after the IR6domain, i.e. this extracellular domain is composed of the IR1, VR1, IR2,VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6 regions of B. garinii (strainpBr).

SEQ ID NO: 3 corresponds to the sequence of the VlsE extracellulardomain of B. garinii (strain pLi) deleted of its signal sequence and ofthe C-terminal region of the mature protein located after the IR6domain, i.e. this extracellular domain is composed of the IR1, VR1, IR2,VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6 regions of B. garinii (strainpLi).

SEQ ID NO: 4 corresponds to the sequence of the VlsE extracellulardomain of B. afzelii (strain pKo) deleted of its signal sequence and ofthe C-terminal region of the mature protein located after the IR6domain, i.e. this extracellular domain is composed of the IR1, VR1, IR2,VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6 regions of B. afzelii (strainpKo).

SEQ ID NO: 5 corresponds to the sequence of the VlsE extracellulardomain of B. burgdorferi sensu stricto (strain B31) deleted of itssignal sequence and of the C-terminal region of the mature proteinlocated after the IR6 domain, i.e. this extracellular domain is composedof the IR1, VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6 regionsof B. burgdorferi sensu stricto (strain B31).

SEQ ID NO: 6 corresponds to the sequence of the IR6 domain of B.burgdorferi sensu stricto (strain B31).

SEQ ID NO: 7 corresponds to the sequence of the IR6 domain of B. afzelii(strain ACA-1).

SEQ ID NO: 8 corresponds to the sequence of the IR6 domain of B. garinii(strain Ip90).

SEQ ID NO: 9 corresponds to the sequence of the VR6 variable region ofB. burgdorferi sensu stricto (strain B31). This sequence can beintroduced into the construct as a spacer arm between the IR6 domains.

It is possible to add a sequence of at least 6 histidines (polyhistidinetail), identified in SEQ ID NO: 10, encoded by any one of the nucleicsequences identified in SEQ ID NOs 11, 12 and 13, at the N-terminal orC-terminal end of the protein in order to allow its purification onmetal-chelate resin, and also additional amino acids represented in SEQID NO: 14 and encoded by the sequence SEQ ID NO: 15, upstream of thepolyhistidine tail. In this configuration, the protein comprises orconsists of a sequence identified as SEQ ID NO: 21. Alternatively, it ispossible to place a sequence of 8 histidines, represented in. SEQ ID NO:16 and encoded by SEQ ID NO: 17, in the N-terminal position of theprotein in place of the 6-histidine sequence, which makes it possible tostabilize the attachment of the recombinant protein to the metal-chelateresin and to improve the purification conditions, and also additionalamino acids represented in SEQ ID NO: 18 and encoded by the sequence SEQID NO: 19. In this configuration, the protein comprises or consists of asequence identified as SEQ ID NO: 23.

The preferred proteins of the invention are those identified as SEQ IDNOs: 21 and 23, respectively encoded by the sequences SEQ ID NOs: 22 and24.

The subject of the invention is also the DNA sequences encoding theproteins as defined above, and in particular the sequences identified asSEQ ID NOs: 22 and 24.

The subject of the invention is also an expression cassette which isfunctional in a cell derived from a prokaryotic organism (example:Escherichia coli) or a eukaryotic organism, such as a yeast (example:Pichia, Schizosaccharomyces), allowing the expression of the nucleicacid described above (DNA), when it is placed under the control of theelements allowing its expression, and also the vector comprising such acassette.

The protein of the invention can in particular be used for the diagnosisof a Borrelia infection. Thus, the subject of the present invention is amethod for the in vitro diagnosis of Lyme borreliosis in a biologicalsample (for example a serum, blood, plasma, etc., sample), according towhich the biological sample is brought into contact with at least oneprotein as defined above and it is determined whether there is formationof an immunological complex between said protein and antibodies of thebiological sample (IgGs and/or IgMs), for example by adding at least oneanti-human-immunoglobulin labeled with any appropriate label. The term“label” is intended to mean a tracer capable of generating a signal. Anonlimiting list of these tracers comprises enzymes which produce asignal detectable, for example, by colorimetry, fluorescence orluminescence, for instance horseradish peroxidase, alkaline phosphatase,β-galactosidase or glucose-6-phosphate dehydrogenase; chromophores, forinstance fluorescent, luminescent or coloring compounds; electron densegroups that can be detected by electron microscopy or via theirelectrical properties, for instance conductivity, by amperometry orvoltammetry methods, or by impedance measurements; groups that can bedetected by optical methods, for instance diffraction, surface plasmonresonance or contact angle variation, or by physical methods, forinstance atomic force spectroscopy, tunnel effect, etc.; radioactivemolecules, for instance ³²P, ³⁵S or ¹²⁵I. Preferably, the protein isimmobilized on a solid support which may be the tip of a Vidas®apparatus, the wells of a microtitration plate, a particle, a gel etc.

In one embodiment of the invention, the sample is also brought intocontact with at least one chimeric fusion protein selected from thosedescribed below:

(a) a protein of which the amino acid sequence comprises (or consistsof) the sequence SEQ ID NO: 25 and the sequence SEQ ID NO: 26 or asequence which exhibits at least 40% identity with SEQ ID NO: 25 and asequence which exhibits at least 50% identity with SEQ ID NO: 26,(b) a protein of which the amino acid sequence comprises (or consistsof) the sequence SEQ ID NO: 27 and the sequence SEQ ID NO: 28 or asequence which exhibits at least 40% identity with SEQ ID NO: 27 and asequence which exhibits at least 50% identity with SEQ ID NO: 28,(c) a protein of which the amino acid sequence comprises (or consistsof) a sequence selected from:(i) the sequence SEQ ID NO: 29 and the sequence SEQ ID NO: 31 or asequence which exhibits at least 40% identity with SEQ ID NO: 29 and asequence which exhibits at least 50% identity with SEQ ID NO: 31,(ii) the sequence SEQ ID NO: 30 and the sequence SEQ ID NO: 31 or asequence which exhibits at least 40% identity with SEQ ID NO: 30 and asequence which exhibits at least 50% identity with SEQ ID NO: 31,(iii) the sequence SEQ ID NO: 29, the sequence SEQ ID NO: 30 and thesequence SEQ ID NO: 31, or a sequence which exhibits at least 40%identity with SEQ ID NO: 29, a sequence which exhibits at least 40%identity with SEQ ID NO: 30 and a sequence which exhibits at least 50%identity with SEQ ID NO: 31,(d) a protein of which the amino acid sequence comprises (or consistsof) a sequence selected from SEQ ID NOs: 32, 34, 36 or a sequenceselected from SEQ ID NOs: 33, 35, 37 and 38 described in greater detailbelow.

Each of the proteins identified above comprises at least one sequence ofthe extracellular domain of a DbpA protein of a Borrelia speciesselected from B. afzelii (SEQ ID NO: 25), B. burgdorferi sensu stricto(SEQ ID NO: 27) and B. garinii (group III: SEQ ID NO: 29) (group IV: SEQID NO: 30) or a sequence exhibiting at least 40% identity with saidsequences, and at least one sequence of an OspC protein of B. afzelii(SEQ ID NO: 26), B. burgdorferi sensu stricto (SEQ ID NO: 28) and B.garinii (SEQ ID NO: 31) or a sequence which exhibits at least 50%identity with said sequences. Preferentially, the DbpA sequence(s) is(are) placed on the N-terminal side of the recombinant protein and theOspC sequence is placed on the C-terminal side of the recombinantprotein.

As described previously, a sequence of at least 6 histidines can beadded at the N-terminal or C-terminal end of the protein in order toenable its purification on metal-chelate resin. The 6-histidinesequence, identified in SEQ ID NO: 10, is preferentially placed on theN-terminal side of the construct. Additional amino acids may be presentupstream of the poly-His tail owing to the insertion, into the codingDNA sequence, of a small sequence which makes it possible to facilitatethe cloning of the sequence of interest into the expression plasmid, forexample the “MRGS” motif (SEQ ID NO: 14) encoded by ATGAGGGGATCC (SEQ IDNO: 15).

A linking region can be introduced between each of the DbpA and OspCsequences which makes up a chimeric recombinant protein. This type ofregion corresponds to a flexible spacing region providing betteraccessibility of the potential antibodies to each of the domains. It isgenerally rich in Gly and Ser amino acids, which are amino acidsdescribed as providing flexibility in the tertiary structure of theprotein. It is also possible to introduce, into a coding sequence ofinterest, a DNA arm (or linker) in order to promote the linking betweenthe coding sequences for two proteins of interest. This is, for example,the “GSGG” motif (SEQ ID NO: 46) encoded by sequence GGTTCCGGGGGT (SEQID NO: 47), which acts as a linker arm between the DbpA group IV andOspC proteins of B. garinii.

Examples of these proteins are represented by SEQ ID NOs: 33, 35, 37 and38 in the sequence listing.

The proteins described above and identified as SEQ ID NOs: 32 to 38 inthe sequence listing are respectively encoded by the corresponding DNAsequences identified in SEQ ID NOs: 39 to 45.

The subject of the invention is also a kit for the in vitro diagnosis ofLyme borreliosis comprising at least one VlsE chimera protein asdescribed above and optionally at least one DbpA/OspC chimeric fusionprotein as defined previously, and preferably comprising at least oneanti-human-immunoglobulin labeled with any appropriate labelcorresponding to the definitions given previously.

EXAMPLES Example 1 Preparation of Plasmid Constructs Encoding the VlsEChimeric Recombinant Proteins

The DNA sequences encoding the various sequences of the protein areidentified in table 1.

TABLE 1 Sequence origin B. burgdorferi species protein B. sensu strictoB. afzelii B. garinii VlsE — — *PBi; **aa 20-293; ***AJ630106 (GenScriptCorp) IR6 *B31; *ACA-1; *Ip90; **aa 167-191; **aa 274-305; **aa 172-188;***AAN87834 ***U76405 ***U76405 (GeneArt GmbH) (GeneArt GmbH) (GeneArtGmbH) *Isolate; **amino acids (aa); ***GenBank accession No.

The sequences were optimized for their expression in E. coli usingGeneOptimizer™ and synthesized respectively by GenScript corporation(Scotch Plains, N.J., USA) or GeneArt GmbH (Regensburg, Germany).

Additional modifications to the DNA, deletions or combinations ofvarious sequences were carried out by PCR by genetic engineering usingthe PCR techniques well known to those skilled in the art and described,for example, in Sambrook J. et al., Molecular Cloning: A LaboratoryManual, 1989. The DNA sequences were ligated into the pMR [2] or pET-3d(Novagen®) expression vector. The plasmid constructs and thecorresponding proteins cited as example (bLYM110, bLYM125) are describedin table 2.

TABLE 2 Plasmid constructs and corresponding proteins Plasmid constructcharacteristics Recombinant protein characteristics Site of insertion ofN-terminal Parental the insert sequence Name Tag B. burgdorferi sequencevector into the vector bLYM110 6 x His VlsE garinii pBi aa 20-293 +pMR78 5′BamHI/3′HindIII SEQ ID 3 IR6 [sensu stricto B21 aa NO: 21274-305 + afzelii ACA-1aa bLYM125 8 x His 172-188 + pET-3d5′NcoI/3′BamHI SEQ ID garinii Ip90 aa 167-191] NO: 23

Example 2 Expression of the Recombinant Proteins of Example 1 andPurification

A plasmid construct described in example 1 was used to transform an E.coli bacterium (strain BL21) according to a conventional protocol knownto those skilled in the art. The transformed bacteria were selected byvirtue of their ampicillin resistance carried by the pMR or pET vector.

A clone of a recombinant bacterium was then selected in order toinoculate a preculture of 40 ml of 2×YT medium (16 g/l tryptone; 10 g/lyeast extract; 5 g/l NaCl, pH 7.0) containing 100 μg/ml ampicillin.After 15 to 18 hours of incubation at 30° C. with shaking at 250 rpm,this preculture was used to inoculate 1 liter of 2×YT medium containing2% glucose and 100 μg/ml ampicillin. This culture was incubated at 30°C. with shaking at 250 rpm until the OD at 600 nm reaches 1.0/1.2. Theculture was maintained for 3 hours 30 min. or 4 hours at 30° C. whileadding 0.4 mM isopropyl-β-D-thio-galactopyranoside (IPTG) and harvestedby centrifugation at 6000 g for 30 min. The cell pellet was stored at−60° C. For the purification, the wet biomass was resuspended in a lysisbuffer containing protease inhibitors without EDTA (Roche) and benzonasenuclease (Novagen®), and subjected to cell rupture at 1.6 kBar in a celldisrupter (Constant Systems Ltd, Daventry, United Kingdom). The lysatewas then centrifuged at 10 000 rpm for 45 minutes at 2-8° C. Afterfiltration through a 0.22 μm filter, the supernatant was loaded onto anNi-NTA column (Qiagen®) equilibrated in a lysis buffer. The resin wasthen washed with the same buffer until the A_(280 nm) reached the baseline. An elution was carried out with the elution buffer, and thepurified protein was dialyzed in a Pierce Slide-A-Lyser® 10000 or 20000MWCO dialysis cassette against the dialysis buffer. The conditions forpurification on Ni-NTA gel are described in table 3.

TABLE 3 Recombinant protein purification bLYM110 bLYM125 Protein SEQ IDNO: 21 SEQ ID NO: 23 Lysis and washing buffer Buffer A ¹ Buffer A ¹ + 2Murea Elution buffer Buffer B ² Buffer B ² modified with 600 mM imidazoleElution step 1 86% Buffer A + 14% 92% Buffer A + 8% Buffer B (4CV)Buffer B (4CV) Elution step 2 100% Buffer B 100% Buffer B Purificationyield 0.5 0.8 mg protein/g wet biomass Purification yield 8.7 17 mgprotein/L of culture ¹ 50 mM sodium phosphate, 30 mM imidazole, 500 mMNaCl, 0.1% Tween 20, 5% glycerol, pH = 7.8 ² 50 mM sodium phosphate, 325mM imidazole, 500 mM NaCl, 5% glycerol, pH = 7.5

The samples were analyzed on NuPAGE® Novex® 4-12% in a NuPAGE® MES-SDScirculating buffer, according to the instructions of the producer(Invitrogen™). The proteins were either stained with Coomassie brilliantblue or were transferred electrophoretically onto a nitrocellulosemembrane. The membrane was blocked with 5% (w/v) dry milk in PBS andincubated with an anti-pentahistidine antibody (Qiagen®) in PBScontaining 0.05% Tween 20. A horseradish peroxidase-labeled goatanti-mouse IgG conjugate (Jackson Immunoresearch laboratories) inPBS/Tween was used as secondary antibody.

The protein concentration was determined using the Bradford Assay Kit(Pierce Coomassie Plus, Perbio Science) with BSA as protein standard.

Example 3 Detection of Human IgGs and IgMs with the Chimeric RecombinantProtein bLYM110 of Example 2 Using a Line Immunoblot Technique

The recombinant protein was deposited onto a polyvinylidene difluoridemembrane (PVDF, Immobilon, Millipore®, Bedford, Mass. USA) according tothe following protocol:

The protein concentration was adjusted to 1 mg/ml in PBS, pH 7.2, anddiluted in PBS, pH 7.2, supplemented with 0.03% Tween 20 (dilution1/200^(th)). The PVDF membrane was wetted in methanol, washed indemineralized water and laid out on a wet blotting paper. A plasticruler was immersed in the protein dilution and attached to the PVDFmembrane. After depositing of the proteins and drying of the membranes,the membranes were cut vertically into narrow strips. Before use, thenarrow strips were incubated with 5% gelatin in TBS, pH 7.5, for 1 hourat 37° C. The immunoblot protocols were carried out at ambienttemperature as described by Bretz A. G. et al. [3]. The narrow stripswere incubated for 2 hours with human sera diluted to 1/200^(th) in TBSwith 1% gelatin, washed and incubated with anti-human IgGs or IgMslabeled with alkaline phosphatase (Sigma™, St-Louis, USA) diluted to1/1000^(th) in TBS with 1% gelatin. After washing, the narrow stripswere incubated with the BCIP-NBT substrate (KPL, Gaithersburg, Md., USA)for 30 minutes, washed in distilled water and dried.

Panel of Sera Tested

The human sera were collected from clinically well-defined, typical LBpatients corresponding to the various stages of LB (22 with erythemamigrans [EM], 5 with carditis, 20 with neuroborreliosis [NB], 20 withLyme arthritis [LA], 20 with acrodermatitis chronica atrophicans [ACA]and 10 with lymphadenosis cutis benigna [LCB]). Anti-Lyme IgGs werefound by immunoblot, described previously using whole cell lysates [4],in the sera of patients with LA, ACA and carditis. EM, NB and LCB wereidentified clinically, but not all the corresponding sera were found tobe positive using the immunoblot [4], or using the commerciallyavailable kits (Vidas® Lyme (Biomeriéux®), Borrelia IgG (Diasorin®) andBorrelia IgM (r-Biopharme®). On the other hand, all the cases of NBincluded in the study had detectable antibodies in the cerebrospinalfluid [CSF] (index extending from 2 to 27.1).

The negative control group consisted of 31 sera previously found to benegative for the presence of anti-Lyme antibodies in conventionalassays. Furthermore, 64 sera from healthy blood donors residing in aregion endemic for Lyme disease (Monthley, Valis, Switzerland) weretested with the recombinant protein. The strength of the reaction wasevaluated as follows: [+], [++], [+++], [−] or equivocal results. Theequivocal results were considered to be negative.

The results are given in table 4 below.

TABLE 4 Stage I Stage II Stage III Donors IgG EM NB Carditis LA ACALymph. (n = 64) (n = 22) (n = 20) (n = 5) (n = 19) (n = 20) (n = 10) 1720 5 19 20 9 6 77.3% 100% 100% 100% 100% 90% 9.4% 12 [+++] 11 [+++]  4[+++] 13 [+++] 20 [+++]  3 [+++] 6 [+] 4 [++] 7 [++] 1 [++] 4 [++] 2[++] 1 [+]  2 [+]  2 [+]  4 [+]  Total IgG positives 93.7% IgM EM NBCarditis (n = 64) (n = 22) (n = 20) (n = 5)  5  4 2 1  22%  20%  40%1.5%  1 [++]  2 [++] 1 [++] 1 [+] 4 [+] 1 [+] 1 [+]  Total IgM positives23.4%

IgG Detection

The results indicate that the recombinant protein bLYM110 is adiagnostic antigen that is highly sensitive at all stages of theinfection for IgGs. At stage I of the infection, the IgGs were detectedin 17 cases of patients with EM out of 22 (i.e. 77.3% sensitivity). Fiveof the patients with EM who are found to be negative with therecombinant protein are also found to be negative with the in-houseimmunoblot and with the commercially available kits. Seven EM sera foundto be positive with the recombinant protein were not detected byimmunoblot, which represents a 31.8% improvement in sensitivity with therecombinant protein. At the primary stage of the infection, in theabsence of characteristic redness, the diagnosis can be difficult sincethe other clinical manifestations of Lyme disease are not specific.Furthermore, only a few patients with EM are detected using theconventional tests. Therefore, the protein of the invention improves thedetection of IgGs at stage I of the infection, bringing their detectionto more than 77% in patients with EM.

IgM Detection

Anti-chimera protein IgMs are found in 23.4% of the LB sera. The proteindetects the IgGs more often than the IgMs in the sera of stage-I and -IILB patients.

Example 4 Preparation of the Plasmid Constructs Encoding the DpbA-OspCChimeric Recombinant Proteins

The DNA sequences encoding the various DpbA and OspC sequences describedare identified in table 5. The DNA sequences were optimized in order topromote expression in E. coli using GeneOptimizer™ and synthesizedrespectively by GenScript corporation (Scotch Plains, N.J., USA) orGeneArt GmbH (Regensburg, Germany).

TABLE 5 Sequence origin B. burgdorferi species protein B. sensu strictoB. afzelii B. garinii DbpA *B31; **aa 2-192; *PKo; **aa 2-150; *40; **aa2-187; ***AF069269 ***AJ131967 ***AF441832 *PBi; **aa 2-176; ***AJ841673OspC *B31; **aa 26-210; *PKo; **aa 2-212; *PEi; **aa 32-208; ***X73622***X62162 ***AJ749866 *Isolate; **amino acids (aa); ***GenBank accessionNo.

Each chimeric recombinant protein comprises at least one epitope regioncorresponding to the extracellular domain of a DbpA sequence of Borreliaburgdorferi sensu stricto or B. afzelii or B. garinii and at least oneepitope region corresponding to the extracellular domain of an OspCsequence of Borrelia burgdorferi sensu stricto or B. afzelii or B.garinii.

The combinations of various nucleotide sequences encoding DbpA and/orOspC sequences and also the modifications of nucleotide sequences, suchas deletions, addition of a linking sequence or addition of a linkersequence, were carried out by genetic engineering using the PCRtechniques well known to those skilled in the art and described, forexample, in Sambrook J. et al., Molecular Cloning: A Laboratory Manual,1989.

The DNA sequences encoding the chimeric proteins of interest wereintroduced into the pMR expression vector [2] between the BamHIrestriction site in the 5′ position and the EcoRI or HindIII site in the3′ position. The plasmid constructs and the corresponding proteins citedas example (bLYM114, bLYM120 and bLYM121) are described in table 6. Thepresence of MRGS in the N-terminal position of the recombinant proteinsand the corresponding nucleotide sequence ATG AGG GGA TCC was introducedby the cloning technique used into the pMR expression vector. Only theATG start codon and consequently the Met amino acid are really essentialin this sequence.

A poly-His sequence (6×histidine) was introduced on the N-terminal sideof each recombinant protein. This sequence allows purification of therecombinant proteins on a metal-chelate affinity column. It is a regionfor attachment to the Ni-NTA gel which makes it possible to subsequentlyfacilitate the step of purifying the chimeric recombinant protein. ThisHHHHHH peptide (SEQ ID NO: 10) is encoded by the nucleotide sequencesCATCATCATCATCATCAT (SEQ ID NO: 11) or CATCATCATCATCATCAC (SEQ ID NO: 12)or CATCATCACCACCATCAT (SEQ ID NO: 13) or by any other sequence encodingthe sequence SEQ ID NO: 10.

By way of indication, this particular attachment region, comprising asuccession of histidines, allows in particular the oriented attachmentof the recombinant protein to a support consisting of silica or of metaloxides.

TABLE 6 Plasmid constructs and corresponding proteins Plasmid constructcharacteristics Recombinant protein characteristics Site of insertion ofN-terminal Parental the insert sequence Name Tag B. burgdorferi sequencename vector into the vector bLYM114 6 x His B. afzelii strain PKo pOL114pMR78* 5′BamHI/3′EcoRI SEQ ID DbpA aa 2-150 + NO: 33 OspC aa 2-212bLYM120 6 x His B. sensu stricto strain B31 pOL120 pMR78*5′BamHI/3′HindIII SEQ ID DbpA aa 28-192 + NO: 35 OspC aa 26-210 bLYM1216 x His B. garinii pOL121 pMR78* 5′BamHI/3′HindIII SEQ ID DbpA III aa25-187 strain NO: 38 40 + DbpA IV aa 24-176 strain PBi + OspC aa 32-208strain PEi

Example 5 Expression of the Recombinant Proteins bLYM114, bLYM120 andbLYM121 of Example 4 and Purification

A plasmid construct in which a sequence SEQ ID NO: 40, 42 or 45 has beeninserted into an expression vector (pMR) was used to transform an E.coli bacterium (strain BL21) according to a conventional protocol knownto those skilled in the art. The transformed bacteria were selected byvirtue of their ampicillin resistance carried by the pMR vector.

A clone of a recombinant bacterium was then selected in order toinoculate a preculture of 40 ml of 2×YT medium (16 g/l tryptone; 10 g/lyeast extract; 5 g/l NaCl, pH 7.0) containing 100 μg/ml of ampicillin.After 15 to 18 hours of incubation at 30° C. with shaking at 250 rpm,this preculture was used to inoculate 1 liter of 2×YT medium containing2% glucose and 100 μg/ml of ampicillin. This culture was incubated at30° C. with shaking at 250 rpm until the OD at 600 nm reaches 1.0/1.2.The culture was maintained for 3 hours 30 min. or 4 hours at 30° C.while adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG), andharvested by centrifugation at 6000 g for 30 min. The cell pellet wasstored at −60° C. For the purification, the wet biomass was thawed andresuspended in a lysis buffer containing protease inhibitors withoutEDTA (Roche™) and benzonase nuclease (Novagen), and subjected to cellrupture at 1.6 kBar in a cell disruptor (Constant Systems Ltd, Daventry,United Kingdom). The lysate was then centrifuged at 10 000 rpm for 45min. at 2-8° C. The supernatant obtained contains the soluble proteins.This supernatant was filtered through a 0.45μ filter and purified byaffinity chromatography on a metal chelation column(nickel-nitrilotriacetic acid matrix (Ni-NTA, Qiagen)). To do this, thesupernatant was loaded (1 ml/min) at 18-25° C. onto an 8 ml column ofNi-NTA gel equilibrated in buffer A (see table 7). The column was thenwashed in buffer A, until an OD_(280 nm)=0 was obtained at the columnoutlet. The elution of the recombinant protein is obtained by applying abuffer B, according to the indications reported in table 7, and thepurified protein was dialyzed in a 10000 ou 20000 MWCO dialysis cassette(Slide-A-Lyser®, Pierce) against a dialysis buffer. The conditions forpurification on Ni-NTA gel are described in table 7.

TABLE 7 Recombinant protein purification Protein bLYM114 bLYM120 bLYM121Lysis and washing Buffer A ¹ buffer Elution buffer Buffer B ² Elutionstep 1 90% Buffer A + 92% Buffer A + 100% 10% Buffer B (4CV) 8% Buffer B(4CV) Buffer B Elution step 2 100% Buffer B 100% Buffer B NAPurification yield 12  13  20 mg protein/g wet biomass Purificationyield 80 122 245 mg protein/L of culture ¹ 50 mM sodium phosphate, 30 mMimidazole, 500 mM NaCl, 0.1% Tween 20, 5% glycerol, pH = 7.8 ² 50 mMsodium phosphate, 325 mM imidazole, 500 mM NaCl, 5% glycerol, pH = 7.5

The samples were analyzed on NuPAGE® Novex® 4-12% in a NuPAGE® MES-SDSbuffer, according to the instructions of the producer (Invitrogen). Theproteins were either stained with Coomassie brilliant blue or weretransferred electro-phoretically onto a nitrocellulose membrane. Themembrane was blocked with 5% (w/v) dry milk in PBS and incubated with anantipentahistidine antibody (Qiagen®) in PBS containing 0.05% Tween 20.A horseradish peroxidase-labeled goat anti-mouse IgG conjugate (JacksonImmunoresearch laboratories) in PBS/Tween was used as secondaryantibody.

The protein concentration was determined using the Bradford kit (PierceCoomassie Plus, Perbio Science) with BSA as protein standard.

Example 6 Detection of Human IgGs and IgMs with the Chimeric RecombinantProteins Using a Line Immunoblot Technique

Each recombinant protein was deposited on a polyvinylidene difluoridemembrane (PVDF, Immobilon, Millipore, Bedford, Mass. USA) according tothe following protocol:

The protein concentration was adjusted to 1 mg/ml in PBS, pH 7.2, anddiluted in PBS, pH 7.2, supplemented with 0.03% Tween 20 (dilution1/200^(th)). The PVDF membrane was wetted in methanol, washed indemineralized water and laid out on a wet blotting paper. A plasticruler was immersed in the protein dilution and attached to the PVDFmembrane. After depositing of the proteins and drying of the membranes,the membranes were cut vertically into narrow strips. Before use, thenarrow strips were incubated with 5% gelatin in TBS, pH 7.5, for 1 hourat 37° C. The immunoblot protocols were carried out at ambienttemperature as described by Bretz A. G. et al. [3]. The narrow stripswere incubated for 2 hours with human sera diluted to 1/200^(th) in TBSwith 1% gelatin, washed and incubated with an anti-human-IgG oranti-human-IgM antibody labeled with alkaline phosphatase (Sigma,St-Louis, USA) diluted to 1/1000^(th) in TBS with 1% gelatin. Afterwashing, the narrow strips were incubated with the alkaline phosphatasesubstrate BCIP-NBT (KPL, Gaithersburg, Md., USA) for 30 min., and thenwashed in distilled water and dried.

Panel of Sera Tested

The human sera were collected from clinically well-defined, typical LBpatients corresponding to the various stages of LB (22 with erythemamigrans [EM], 5 with carditis, 20 with neuroborreliosis [NB], 20 withLyme arthritis [LA], 20 with acrodermatitis chronica atrophicans [ACA]and 10 with lymphadenosis cutis benigna [LCB]). Anti-Lyme IgGs werefound by immunoblot, using whole cell lysates [4], in the sera ofpatients with LA, ACA and carditis. EM, NB and LCB were identifiedclinically, but not all the corresponding sera were found to be positiveby immunoblot [4], or using the commercially available kits (Vidas® Lyme(biomérieux), Borrelia IgG (Diasorin®) and Borrelia IgM (r-Biopharm®)).On the other hand, all the cases of NB included in the study haddetectable antibodies in the cerebrospinal fluid [CSF] (index extendingfrom 2 to 27.1 with Vidas® Lyme (biomérieux)). The presence of IgM wassought only in the stage I and stage II clinical cases and not in thechronic stages.

The negative control group consisted of 31 sera previously found to benegative for the presence of anti-Lyme antibodies in conventionalassays. Furthermore, 64 sera from healthy blood donors residing in aregion endemic for Lyme disease (Monthley, Valais, Switzerland) weretested with the recombinant protein.

The strength of the reaction was evaluated as follows: [+], [++], [+++],[−] or equivocal results. The equivocal results were considered to benegative.

The results are given in table 8 below.

TABLE 8 Reactivity in Line immunoblot of human sera from patients withLyme borreliosis, with 3 chimeric recombinant proteins IgG IgM Stage IStage II Stage III Stage I Stage II Recombinant EM NB Carditis LA ACALCB EM NB Carditis protein (n = 22) (n = 20) (n = 5) (n = 19) (n = 20)(n = 10) (n = 22) (n = 20) (n = 5) bLYM114 5 10 0 7 12  2  7 7 2 bLYM1206  7 0 8 6 0 11 7 2 bLYM121 2 10 5 9 8 0  7 7 2 Σ bLYM 9 13 5 18  17  211 7 2 114 + 120 + 121 Positive 40.9% 59.1% 100% 94.7% 85% 20% 50% 35%40% sera (%) and  1 [+++]  8 [+++]  4 [+++]  7 [+++]  8 [+++] 1 [++]  1[+++] 5 [++] 2 [++] reaction 4 [++] 2 [++] 1 [+] 8 [++] 5 [++] 1 [+]  7[++] 2 [+]  strength 4 [+]  3 [+]  3 [+]  4 [+]  5 [+]  Total 66.7%positives  28 [+++] and reaction 20 [++] strength 16 [+]  42.5%  1 [+++]14 [++] 7 [+]

The specificity is 100% on the basis of 31 sera originating from healthyindividuals determined to be Lyme-negative using the standardcommercially available tests.

IgG Detection

The results indicate that the recombinant chimeric fusion proteins arediagnostic tools that are sensitive at all stages of the infection forIgGs and IgMs. They demonstrate an additional effect of the threerecombinant proteins based, respectively, on sequences of Borreliaafzelii, B. sensu stricto and B. garinii for the detection of IgGs. Thecombined use of the three chimeric recombinant proteins makes itpossible, at stage I of the infection, to detect IgGs in 9 cases ofpatients with EM out of 22 (i.e. 40.9% sensitivity).

IgM Detection

Anti-chimera protein IgMs are found in 11 cases out of 22 (i.e. 50%sensitivity). These chimera proteins therefore detect the IgMs moreoften than the IgGs in the sera of stage-I LB patients. The testsperformed as a control: immunoblot [4], and commercially available kitBorrelia IgM (r-Biopharm®) do not further detect IgM-positive sera. Inaddition, 3 sera found to be negative using the immunoblot test andBorrelia IgM (r-Biopharm®) are detected by the three chimeric proteinscited as example (3/3) or by one of the three proteins cited as example(1/3). The combined use of the three recombinant proteins makes itpossible to improve the IgM detection sensitivity by 13.6% in stage I ofthe infection.

Example 7 Evaluation and Validation of the Chimeric Recombinant ProteinsbLYM114, bLYM120, bLYM121 and bLYM125 in a VIDAS® test (bioMérieux)

This validation is carried out in a VIDAS® test using:

1) the recombinant chimeric proteins bLYM114, bLYM120 and bLYM121,obtained according to examples 4 and 5 for IgM detection, and

2) the chimeric recombinant proteins bLYM114 and bLYM120, obtainedaccording to examples 4 and 5 and the chimeric protein bLYM125, obtainedaccording to examples 1 and 2, for the IgG detection.

The principle of the VIDAS° test is the following: a tip constitutes thesolid support which also serves as a pipetting system for the reagentspresent in the strip. The recombinant protein(s) is (are) attached tothe tip. After a dilution step, the sample is drawn up and forced backseveral times in the tip. This allows the anti-Lyme immunoglobulins inthe sample to bind to the recombinant proteins. The unbound proteins areremoved by washing. An anti-human-immunoglobulin antibody conjugated toalkaline phosphatase (ALP) is incubated in the tip, where it binds tothe anti-Lyme immunoglobulins. Washing steps remove the unboundconjugate. During the final visualizing step, the alkaline phosphatase(ALP) substrate, 4-methylumbelliferyl phosphate, is hydrolyzed to4-methyl-umbelliferone, the fluorescence of which emitted at 450 nm ismeasured. The intensity of the fluorescence is measured by means of theVidas® optical system and is proportional to the presence of anti-Lymeimmunoglobulins present in the sample. The results are analyzedautomatically by the VIDAS® and expressed as RFV (Relative FluorescentValue).

255 positive sera (equivocal sera+positive sera) and 298 negative sera(equivocal+negative) were thus assayed with the Vidas® system.

The Vidas® Lyme IgG tips are sensitized with 300 μL of solutioncomprising the bLYM114, bLYM120 and bLYM125 proteins of the invention,each at a concentration of 1 pg/mL in a common sensitizing solution.

In the first step, the sera are incubated for 5.3 min. for the formationof the antigen-antibody complexes. In the second step, anti-human-IgGslabeled with ALP are incubated for 5.3 min.

The results are given as an index relative to a positivity thresholdpositioned at 135 RFV in the protocol.

Among the 255 positive sera tested, 246 are positive and 9 are falselynegative, which corresponds to a sensitivity of 96.5%.

Among the 298 negative sera tested, 284 are negative and 14 are falselypositive, which corresponds to a specificity of 95.3%.

LITERATURE REFERENCES

-   1. Göttner G. et al., Int. J. Microbiol. 293, Suppl. 37, 172-173    (2004)-   2. Arnaud N. et al., Gene 1997; 199:149-156.-   3. Bretz A. G., K. Ryffel, P. Hutter, E. Dayer and O. Péter.    Specificities and sensitivities of four monoclonal antibodies for    typing of Borrelia burgdorferi sensu lato isolates. Clin. Diag. Lab.    Immunol. 2001; 8: 376-384.-   4. Ryffel K., Péter O., Rutti B. and E. Dayer. Scored antibody    reactivity by immunoblot suggests organotropism of Borrelia    burgdorferi sensu stricto, B. garinii, B. afzelii and B. valaisiana    in human. J. Clin. Microbiol. 1999; 37:4086-92-   5. Steere A C. et al., Clin Infect Dis 2008; 47:188-195.

1. A Borrelia chimeric protein comprising at least one sequence of anextracellular domain of a VlsE protein of a first Borrelia speciescorresponding to a determined strain and at least one sequence of an IR6region of a VlsE protein of a second Borrelia species or of the firstBorrelia species but corresponding to a strain different than that ofthe first species, said chimeric protein comprising: the sequence of theextracellular domain of the VlsE protein of the first Borrelia specieswhich is composed of five variable regions VR1, VR2, VR3, VR4, and VR5and of six invariable regions IR1, IR2, IR3, IR4, IR5, and IR6, and saidat least one sequence of the extracellular domain is selected from thegroup consisting of the sequences set forth in SEQ ID NOs: 1, 2, 3, 4,and 5, or a variant of one of said sequences set forth in SEQ ID NOs: 1,2, 3, 4, and 5, said variant exhibiting at least 50% identity with thesequence set forth in SEQ ID NOs: 1, 2, 3, 4, and 5, respectively, onthe condition that said variant is capable of forming an immunologicalcomplex with antibodies produced following a Borrelia infection, andsaid at least one sequence of the IR6 region of the second Borreliaspecies, or of the first Borrelia species but corresponding to a straindifferent from that of the first species, which is selected from thegroup consisting of the sequences set forth in SEQ ID NOs: 6, 7, and 8or a variant of one of said sequences set forth in SEQ ID NOs: 6, 7, and8, said variant exhibiting at least 80% identity with the sequences setforth in SEQ ID NOs: 6, 7, and 8, respectively, on the condition thatthe variant of said sequence is capable of forming an immunologicalcomplex with antibodies produced following a Borrelia infection.
 2. Theprotein as claimed in claim 1, further comprising a VR6 sequence of aBorrelia species identified in SEQ ID NO:
 9. 3. The protein as claimedin claim 1, comprising: the sequence set forth in SEQ ID NO: 1 or avariant of said sequence set forth in SEQ ID NO: 1, said variantexhibiting at least 50% identity with the sequence set forth in SEQ IDNO: 1, the sequence set forth in SEQ ID NO: 6 or a variant of saidsequence set forth in SEQ ID NO: 6, said variant exhibiting at least 80%identity with the sequence set forth in SEQ ID NO: 6, the sequence setforth in SEQ ID NO: 7 or a variant of said sequence set forth in SEQ IDNO: 7, said variant exhibiting at least 80% identity with the sequenceset forth in SEQ ID NO: 7, and the sequence set forth in SEQ ID NO: 8 ora variant of said sequence set forth in SEQ ID NO: 8, said variantexhibiting at least 80% identity with the sequence set forth in SEQ IDNO:
 8. 4. The protein as claimed in claim 3, further comprising thesequence set forth in SEQ ID NO:
 9. 5. The protein as claimed in claim1, comprising the sequence set forth in SEQ ID NO: 1, the sequence setforth in SEQ ID NO: 6, the sequence set forth in SEQ ID NO: 7, and thesequence set forth in SEQ ID NO:
 8. 6. The protein as claimed in claim5, comprising the sequence set forth in SEQ ID NO:
 9. 7. The protein asclaimed in claim 1, comprising a sequence selected from the sequencesset forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:
 23. 8. Anucleic acid encoding a protein as defined in claim
 1. 9. An expressioncassette which is functional in a cell derived from a prokaryotic oreukaryotic organism, configured to express the nucleic acid as definedin claim 8 when placed under control of elements necessary for itsexpression.
 10. A vector comprising the expression cassette as definedin claim
 9. 11. A method for the in vitro diagnosis of Lyme borreliosisin a biological sample, the method comprising: contacting the biologicalsample with at least one protein as defined in claim 1; and determiningwhether there is formation of an immunological complex between saidprotein and antibodies of the biological sample.
 12. The method asclaimed in claim 11, in which the antibodies of the biological sampleare IgGs and/or IgMs.
 13. The method as claimed in claim 11, in whichthe formation of the immunological complex is determined by adding atleast one anti-human-immunoglobulin labeled with an appropriate label.14. The method as claimed in claim 9, in which the protein isimmobilized on a solid support.
 15. A kit for the in vitro diagnosis ofLyme borreliosis comprising at least one protein as defined in claim 1.16. The kit as claimed in claim 15, further comprising at least oneanti-human-immunoglobulin labeled with an appropriate label.